1. Field of the Invention
The present invention relates to the homogeneous sulfation of polysaccharides and semisynthetic derivatives thereof, in particular glycosaminoglycans such as hyaluronic acid and its esters and tetraalkylammonium salts, for the preparation of new biomaterials useful in biomedical, health care, and pharmaceutical applications, and to such biomaterials per se. Such sulfated derivatives exhibit anti-thrombotic activity as evidenced by the lengthening of both the thrombin time and the whole blood clotting time. Moreover, the absence of hemolysis and the growth and shape of endothelial cells placed in contact with such sulfated derivatives indicate that these materials are promising heparin-like compounds.
2. Description of Related Art
Many molecules of biological origin are polyelectrolytes, and their interactions are very important in a wide variety of biochemical reactions. Consequently, synthetic and/or semisynthetic polyelectrolytes have been in use for some time now. These polyelectrolytes mimic the biological characteristics of natural polyelectrolytes, and can have somewhat different characteristics compared to the starting material.
Polyelectrolytes of biological origin include sulfated polysaccharides, and in particular, heparin and its derivatives (D.A. Lane and U. Lindahl, Eds., Heparin Chemical and Biological Propertied, Clinical Applications, Edward Arnold, London), which play an important role in cell-substrate interactions, particularly in the process of viral activity inhibition, in the process of blood coagulation, in lipid removal, etc.
Heparin is the most biologically reactive member of the family of sulfated glycosaminoglycans. It is well known for its antithrombotic and anticoagulant properties. In fact, it is extensively used in the management of cardiovascular diseases and contributes enormously to the success of open heart surgery. Nevertheless, the structure of heparin is not simple and, due to the number of variations, is not entirely known. Commercial heparins consist of a spectrum of 21 heparins (Nader et al. (1974) Biochem. Biophys. Res. Commun. 57:488) ranging in molecular weights from 3,000 to 37,500 in varying anticoagulant activities.
The blood anticoagulant activity of heparin is attributed to structural features, e.g., degree of sulfation, degree of dissociation, particular sequences of COO.sup.-- and SO.sub.--.sub.3 groups, as well as to molecular shape and size. These factors appear to be related to biological activity by virtue of their importance in the ion binding capacity of heparin (Stivala et al. (1967) Arch. Biochem. Biophys. 122:40). By virtue of its highly negatively charged nature, heparin has a strong affinity for cations, and its activity is pH-dependent.
Most of the readily available natural polysaccharides have been sulfated in an attempt to obtain heparin analogues (Hoffman et al. (982) Carbohydrate Res. 2:115; Kindness et al. (1980) Brit. J. Pharmac. 69:675; Horton et al. (1973) Carbohydrate Res. 30:349; Okada et al. (1979) Makromol. Chem. 180:813; Kikuchi et al. (1979) Nippon Kagaku Kaishi 1:127; Manzac et al. (1981) Proc. Third M.I.S.A.O. 5:504), and recently, sulfate, carboxylic, and sulfonate groups were attached to synthetic polymers such as polystyrene (Kanmaugue et al. (1985) Biomaterials 6:297) and polyurethane (Ito et al.(1992) Biomaterials 13:131). The anticoagulant activities of these materials were much lower than that heparin, and were dependent on the type and binding of the substituents, the degree of substitution, and sequences.
Some chemical reactions are known which make it possible to sulfate polysaccharides (WO 88/00211; EP 0 340 628; Nagasawa et al. (1986) Carbohydrate Research 158:183-190), but it has not yet been possible to obtain sulfated polysaccharides which, besides the chemical and chemical-physical characteristics peculiar to such polysaccharides, also possess new characteristics, such as anticoagulant activity.